Kyoung Hoon Kim

rapid green synthesis of silver nanoparticles from Chrysanthemum indicum l and its antibacterial and cytotoxic effects: an in vitro study

The present work reports a simple, cost-effective, and ecofriendly method for the synthesis of silver nanoparticles (AgNPs) using Chrysanthemum indicum and its antibacterial and cytotoxic effects. The formation of AgNPs was confirmed by color change, and it was further characterized by ultraviolet–visible spectroscopy (435 nm). The phytochemical screening of C. indicum revealed the presence of flavonoids, terpenoids, and glycosides, suggesting that these compounds act as reducing and stabilizing agents. The crystalline nature of the synthesized particles was confirmed by X-ray diffraction, as they exhibited face-centered cubic symmetry. The size and morphology of the particles were characterized by transmission electron microscopy, which showed spherical shapes and sizes that ranged between 37.71–71.99 nm. Energy-dispersive X-ray spectroscopy documented the presence of silver. The antimicrobial effect of the synthesized AgNPs revealed a significant effect against the bacteria Klebsiella pneumonia, Escherichia coli, and Pseudomonas aeruginosa. Additionally, cytotoxic assays showed no toxicity of AgNPs toward 3T3 mouse embryo fibroblast cells (25 μg/mL); hence, these particles were safe to use.

Crystal Structure of Lipoate-Protein Ligase A Bound with the Activated Intermediate INSIGHTS INTO INTERACTION WITH LIPOYL DOMAINS

Lipoic acid is the covalently attached cofactor of several multi-component enzyme complexes that catalyze key metabolic reactions. Attachment of lipoic acid to the lipoyl-dependent enzymes is catalyzed by lipoate-protein ligases (LPLs). In Escherichia coli, two distinct enzymes lipoate-protein ligase A (LplA) and lipB-encoded lipoyltransferase (LipB) catalyze independent pathways for lipoylation of the target proteins. The reaction catalyzed by LplA occurs in two steps. First, LplA activates exogenously supplied lipoic acid at the expense of ATP to lipoyl-AMP. Next, it transfers the enzyme-bound lipoyl-AMP to the ϵ-amino group of a specific lysine residue of the lipoyl domain to give an amide linkage. To gain insight into the mechanism of action by LplA, we have determined the crystal structure of Thermoplasma acidophilum LplA in three forms: (i) the apo form; (ii) the ATP complex; and (iii) the lipoyl-AMP complex. The overall fold of LplA bears some resemblance to that of the biotinyl protein ligase module of the E. coli biotin holoenzyme synthetase/bio repressor (BirA). Lipoyl-AMP is bound deeply in the bifurcated pocket of LplA and adopts a U-shaped conformation. Only the phosphate group and part of the ribose sugar of lipoyl-AMP are accessible from the bulk solvent through a tunnel-like passage, whereas the rest of the activated intermediate is completely buried inside the active site pocket. This first view of the activated intermediate bound to LplA allowed us to propose a model of the complexes between Ta LplA and lipoyl domains, thus shedding light on the target protein/lysine residue specificity of LplA.

Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

The N-acetyltransferase arrest defective 1 (ARD1) is an important regulator of cell growth and differentiation that has emerged recently as a critical molecule in cancer progression. However, the regulation of the enzymatic and biological activities of human ARD1 (hARD1) in cancer is presently poorly understood. Here, we report that hARD1 undergoes autoacetylation and that this modification is essential for its functional activation. Using liquid chromatography-tandem mass spectrometry and site-directed mutational analyses, we identified K136 residue as an autoacetylation target site. K136R mutation abolished the ability of hARD1 to promote cancer cell growth in vitro and tumor xenograft growth in vivo. Mechanistic investigations revealed that hARD1 autoacetylation stimulated cyclin D1 expression through activation of the transcription factors beta-catenin and activator protein-1. Our results show that hARD1 autoacetylation is critical for its activation and its ability to stimulate cancer cell proliferation and tumorigenesis.

Crystal Structure of 2-Nitropropane Dioxygenase Complexed with FMN and Substrate: identification of the catalytic base

Nitroalkane compounds are widely used in chemical industry and are also produced by microorganisms and plants. Some nitroalkanes have been demonstrated to be carcinogenic, and enzymatic oxidation of nitroalkanes is of considerable interest. 2-Nitropropane dioxygenases from Neurospora crassa and Williopsis mrakii (Hansenula mrakii), members of one family of the nitroalkane-oxidizing enzymes, contain FMN and FAD, respectively. The enzymatic oxidation of nitroalkanes by 2-nitropropane dioxygenase operates by an oxidase-style catalytic mechanism, which was recently shown to involve the formation of an anionic flavin semiquinone. This represents a unique case in which an anionic flavin semiquinone has been experimentally observed in the catalytic pathway for oxidation catalyzed by a flavin-dependent enzyme. Here we report the first crystal structure of 2-nitropropane dioxygenase from Pseudomonas aeruginosa in two forms: a binary complex with FMN and a ternary complex with both FMN and 2-nitropropane. The structure identifies His152 as the proposed catalytic base, thus providing a structural framework for a better understanding of the catalytic mechanism.

Helicobacter pylori proinflammatory protein up-regulates NF-κB as a cell-translocating Ser/Thr kinase

There has been considerable interest in virulence genes in the plasticity region of Helicobacter pylori, but little is known about many of these genes. JHP940, one of the virulence factors encoded by the plasticity region of H. pylori strain J99, is a proinflammatory protein that induces tumor necrosis factor-alpha and interleukin-8 secretion as well as enhanced translocation of NF-κB in cultured macrophages. Here we have characterized the structure and function of JHP940 to provide the framework for better understanding its role in inflammation by H. pylori. Our work demonstrates that JHP940 is the first example of a eukaryotic-type Ser/Thr kinase from H. pylori. We show that JHP940 is catalytically active as a protein kinase and translocates into cultured human cells. Furthermore, the kinase activity is indispensable for indirectly up-regulating phosphorylation of NF-κB p65 at Ser276. Our results, taken together, contribute significantly to understanding the molecular basis of the role of JHP940 in inflammation and subsequent pathogenesis caused by H. pylori. We propose to rename the jhp940 gene as ctkA (cell translocating kinase A).

Green synthesis of silver nanoparticles using Rheum palmatum root extract and their antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa

Abstract In the present study, silver nanoparticles (AgNPs) were synthesized by using aqueous root extracts of Rheum palmatum and characterized by various spectroscopic methods. The nanoparticles were found to be in hexagonal and spherical shapes. The average particle size was found to be 121u2009±u20092u2009nm with zeta potential values of −21.6u2009mv by dynamic light scattering (DLS) method. Gas chromatography–mass spectrometry (GC-MS) analysis of R. palmatum revealed 35 compounds. The synthesized AgNPs showed significant activity against Staphylococcus aureus and Pseudomonas aeruginosa with IC90 values of 15u2009μg/ml and IC50 values of 7.5u2009μg/ml, respectively. The protein leakage level was high and morphological changes occurred in bacteria treated with AgNPs.

Adipogenic gene expression and fatty acid composition in subcutaneous adipose tissue depots of Angus steers between 9 and 16 months of age

We have demonstrated that among carcass adipose tissue depots, brisket subcutaneous adipose tissue contains the greatest concentration of MUFA and lowest concentration of SFA. Therefore, we hypothesized that brisket subcutaneous adipose tissue depots would exhibit greater adipogenic gene expression over time than other major subcutaneous adipose tissue depots. Four Angus steers, each at 9, 12, 14, and 16 mo of age, were harvested and fresh subcutaneous adipose tissue samples were collected from over the brisket, chuck, rib, loin, sirloin, round, flank, and plate. Relative gene expression for C/EBPβ, PPARγ, carnitine palmitoyltransferase-1 beta (CPT-1β), stearoyl-coenzyme A desaturase (SCD), AMP-activated protein kinase alpha (AMPKα), and G-coupled protein receptor 43 (GPR43) was analyzed by quantitative real-time PCR. Expression of C/EBPβ, PPARγ, and CPT-1β was greatest at 12 to 14 mo of age (all P < 0.0001) and declined to very low abundance by 16 mo of age in all depots. Expression of PPARγ and CPT-1β was greater (P < 0.03) in flank, rib, and sirloin subcutaneous adipose tissues than in brisket and round adipose tissues. The expression of the SCD gene did not differ among the 4 age groups (P = 0.95). The palmitoleic:stearic acid ratio (an estimate of SCD activity) was greater (P < 0.001) in the subcutaneous adipose tissues from brisket, plate, and round than in the loin, rib, and sirloin. Conversely, subcutaneous adipose tissue from the loin, rib, and sirloin had greater (P < 0.001) SCD gene expression than the brisket, plate, and round. In general, subcutaneous adipose tissues with the highest concentration of MUFA and least SFA consistently exhibited the least SCD gene expression and adipogenic gene expression. We conclude that MUFA in the brisket and other depots with large SCD indices were deposited before 9 mo of age, during a time when the subcutaneous adipocytes were highly differentiated.

Dietary Supplementation of l-Arginine and Conjugated Linoleic Acid Reduces Retroperitoneal Fat Mass and Increases Lean Body Mass in Rats

We hypothesized that l-arginine and conjugated linoleic acid (CLA) would have additive effects in decreasing adiposity. Sprague Dawley rats were assigned to the following dietary groups (n = 6/group; 5 wk total): 1) control (2.55% l-alanine plus 1.5% canola oil); 2) arginine (1.25% l-arginine plus 1.5% canola oil); 3) CLA (2.55% l-alanine plus 1.5% CLA); and 4) arginine plus CLA (1.25% l-arginine plus 1.5% CLA). Supplemental amino acids were provided in drinking water and CLA was incorporated into the food pellets. Daily weight gain, food intake, arginine intake, and final body and eviscerated body weights were greater in rats fed supplemental CLA then in rats fed canola oil. The retroperitoneal adipose tissue:body weight ratio was less in rats fed supplemental CLA than in rats fed canola oil, but epididymal adipose tissue, liver, and soleus and extensor digitorum longus muscle weights were unaffected by arginine or CLA. CLA decreased epididymal adipose tissue concentrations of palmitoleic, oleic, and cis-vaccenic acid. CLA and arginine increased palmitate oxidation to CO(2) in epididymal adipose tissue in vitro relative to control rats. Glucose and palmitate incorporation into total lipids in epididymal adipose tissue was lower in rats fed supplemental arginine than in alanine-fed rats. Arginine increased plasma glycerol relative to alanine-fed rats and CLA and arginine independently decreased most serum essential amino acids and alanine, glutamate, glutamine, and ornithine. We conclude that CLA and arginine modulated adipose tissue metabolism by separate, but not additive, effects. Also, CLA and arginine may have depressed muscle protein turnover.

The effect of the form of nitrogen in the diet on ruminal fermentation and the yield of microbial protein in sheep consuming diets of grass silage supplemented with starch or sucrose

Three experiments were conducted to investigate the influence of the type of carbohydrate in the supplement on responses of microbial protein synthesis (MPS, calculated from the urinary output of purine derivatives) in the rumen to dietary addition of ruminal sources of peptides and amino acids in sheep consuming grass silage. In Experiment 1, incubations of rumen liquor in vitro indicated that bacteria fermenting sucrose grew better when ammonia was the only source of added nitrogen than did bacteria utilizing starch. These results led to the hypothesis that bacteria fermenting sucrose might be less dependent on performed amino acids and peptides for their growth than are bacteria fermenting starch. Experiment 2 was designed to test this hypothesis by measuring the response of MPS to inclusion of rapeseed meal, as a source of ruminal amino acids and peptides, in supplements of sucrose or starch given to sheep given a basal diet of grass silage. There was no difference between the carbohydrate treatments in the response of MPS to the inclusion of rapeseed meal, but also, there was no difference in MPS between the sucrose and starch supplements when given without added protein. Experiment 3 examined the effect of the level of starch (0.20 and 0.38 of DM) in the diet on the response of MPS to isonitrogenous dietary additions of urea or casein. The hypothesis tested was that the addition of protein would lead to a greater increase of MPS with the higher level of starch because amylolytic bacteria require preformed peptides and amino acids for maximal growth. However, this was not the case because the response of MPS to inclusion of casein as opposed to urea was greater at the lower level of starch. These results question the generalization that amylolytic bacteria require preformed peptides and amino acids for growth and highlight the need for more information on the influence of diet composition in determining the types of amylolytic bacteria that dominate the ruminal fermentation, there being evidence in the literature that at least one potentially dominant organism, Ruminobacter amylophilus, mainly uses ammonia for growth with only very limited incorporation of organic nitrogen.

Co-culture of bovine muscle satellite cells with preadipocytes increases PPARγ and C/EBPβ gene expression in differentiated myoblasts and increases GPR43 gene expression in adipocytes.

We hypothesized that preadipocyte differentiation would be depressed by differentiating myoblasts, whereas preadipocytes would promote adipogenic gene expression in myoblasts in a co-culture system. We also determined the effects of arginine, a biological precursor of nitric oxide, and/or trans-10, cis-12 conjugated linoleic acid (CLA) on adipogenic gene expression during differentiation of bovine preadipocytes and myoblasts. Bovine semimembranosus satellite cells (BSC) and subcutaneous preadipocytes were isolated from crossbred steers and cultured with 10% fetal bovine serum (FBS)/Dulbeccos modified Eagle medium (DMEM) and 1% antibiotics during the 3-day proliferation period. After proliferation, BSC and preadipocytes were treated for 3 days with 3% horse serum/DMEM and 5% FBS/DMEM with antibiotics, respectively. Media also contained 100 μM oleic acid, 10 μg/ml insulin, 1 μg/ml pioglitazone and 1 μg/ml dexamethasone. Subsequently, the differentiating myoblasts and adipocytes were cultured in their respective media with 5 mM arginine and/or 40 μM trans-10, cis-12 CLA for 4 days. Finally, myoblasts and adipocytes were single- or co-cultured for 2 h singly or in combination. Arginine stimulated SCD gene expression, whereas CLA depressed SCD gene expression in adipocytes and myoblasts (P=.002). Co-culture of adipocytes and myoblasts elicited an increase in C/EBPβ and PPARγ gene expression in differentiated myoblasts (P≤.01) and an increase in GPR43 gene expression in adipocytes (P=.01). Expression of AMPKα and CPT1ß was unaffected by co-culture, although SCD gene expression tended (P=.12) to be depressed by co-culture. These experiments demonstrated that co-culture of adipocytes with myoblasts increased adipogenic gene expression in the myoblastic cells.